Work carried out under this proposal will focus on the characterization of non-native states and unfolding pathways for a series of proteins with varying topology and secondary structure. The methods of molecular dynamics and newly developed approaches of radius of gyration umbrella sampling will be used to investigate non-native states of apomyoglobin, fragment B of staphylococcal protein A, lysozyme and apo-alpha-lactalbumin. Models for the protonation states of titratable side chains which are representative of key pH states for the protein apomyoglobin will be developed and studied - models for the system at neutral pH, pH 4 and lower pH values will be considered. These systems will then be investigated using techniques of equilibrium and non-equilibrium (T-jump) molecular dynamics in explicit aqueous solvent. Similar studies will be carried out for lysozyme, apo-alpha-lactalbumin and protein A. Using recently developed techniques of radius of gyration umbrella sampling, the unfolding pathway followed by each of these protein systems (in explicit solvent) when incremental increases in the radius of gyration are induced by changes in the radius of gyration biasing potential will be examined. This pathway, and the intermediates which develop at intermediate values of radius of gyration, will be compared to experimentally determined pathways and intermediates as well as those we find from equilibrium and T-jump simulations. The initial focus of these studies will be to characterize the unfolding pathway for the small three helical bundle protein, protein A, because of its simplified topology. The folding pathway of this system is currently being studied experimentally using fast kinetic methods and NMR hydrogen exchange.